Calcium Blocks Formation of Apoptosome by Preventing Nucleotide Exchange in Apaf- 1  Qing Bao, Wenyun Lu, Joshua D. Rabinowitz, Yigong Shi  Molecular Cell  Volume 25, Issue 2, Pages 181-192 (January 2007) DOI: 10.1016/j.molcel.2006.12.013 Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 1 Reconstitution of Apoptosome In Vitro (A) Caspase-9 activity was assayed by cleavage of the fluorogenic substrate LEHD-AMC. The cleavage was monitored by fluorescence spectrophotometer. The purity of recombinant Apaf-1 and caspase-9 proteins was shown on the left (SDS-PAGE stained by Coomassie blue). (B) Caspase-9 is activated by the assembled apoptosome, but not by the isolated Apaf-1 protein. For the apoptosome involving the full-length Apaf-1 protein, both cytochrome c and dATP were required for the activation of caspase-9. For the miniapoptosome involving Apaf-1ΔC, only dATP, but not cytochrome c, was required for caspase-9 activation. All error bars are standard deviations. Molecular Cell 2007 25, 181-192DOI: (10.1016/j.molcel.2006.12.013) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 2 Calcium Inhibits the Activation of Caspase-9 by Apoptosome (A) Calcium, but not magnesium or manganese, inhibits the activation of caspase-9 by the apoptosome or the miniapoptosome. This inhibition is specifically relieved by an excess amount of EDTA. A fluorogenic caspase-9 substrate, LEHD-AMC, was used in this assay. (B) A repeat of experiments described in (A) using procaspase-3 (C163A) as the substrate. The full-length Apaf-1 protein was used in this assay. (C) Calcium inhibits the formation of apoptosome and miniapoptosome in a concentration-dependent manner. One micromolar calcium resulted in 20%–30% inhibition of apoptosome assembly, whereas 1 mM led to near complete inhibition. All error bars are standard deviations. Molecular Cell 2007 25, 181-192DOI: (10.1016/j.molcel.2006.12.013) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 3 Calcium Inhibits the Activation of Caspase-9 by Blocking the Assembly of Apoptosome (A) Calcium does not inhibit caspase-9 activation by the preassembled apoptosome or miniapoptosome. “M” denotes Apaf-1 monomer. “O” represents the assembled apoptosome or miniapoptosome. All error bars are standard deviations. (B) Calcium significantly undermines the ability of the full-length Apaf-1 to form an apoptosome in the presence of cytochrome c and dATP. This inhibition was reversed by an excess amount of EDTA. Shown here and in (C) and (D) are chromatograms from gel filtration runs, as monitored by UV absorption at 280 nm. (C) Preincubation with calcium significantly undermines the ability of Apaf-1ΔC to form a miniapoptosome in the presence of dATP. Calcium does not appear to have an apparent effect on the disassembly of the miniapoptosome. (D) Concentration-dependent effect of calcium on the assembly of the miniapoptosome. Compared to the absence of calcium, 1 μM calcium resulted in 25% inhibition of miniapoptosome assembly, whereas 1 mM led to nearly complete inhibition. Molecular Cell 2007 25, 181-192DOI: (10.1016/j.molcel.2006.12.013) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 4 Calcium Inhibits Apoptosome Assembly by Interfering with Nucleotide Exchange in Apaf-1 (A) Calcium directly binds to the miniapoptosome. Shown here is a representative ITC experiment. Analysis of the data gave rise to a dissociation constant of ∼19.3 μM. (B) Calcium interferes with nucleotide exchange in Apaf-1ΔC. Quantitative identification of bound nucleotide in Apaf-1ΔC was determined by LC-MS/MS. See text for detailed description. (C) A time course of nucleotide exchange in Apaf-1ΔC. Apaf-1ΔC was incubated with 1 mM dADP in the absence (blue line) or presence (magenta line) of 1 mM calcium chloride. The sample was examined by LC-MS/MS at various time points. All error bars are standard deviations. (D) Calcium interferes with nucleotide exchange in the full-length Apaf-1. Quantitative identification of bound nucleotide in the full-length Apaf-1 protein was determined by LC-MS/MS. Only in the absence of calcium could the majority of bound ADP in the full-length Apaf-1 be replaced by dADP. Molecular Cell 2007 25, 181-192DOI: (10.1016/j.molcel.2006.12.013) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 5 Calcium Binding Has a Direct Consequence on the Conformation of Apaf-1 (A) A representative SDS-PAGE gel showing the effect of calcium on trypsin digestion of the full-length Apaf-1. Note the persistence of the 40 kDa fragment only in the presence of calcium. (B) A representative SDS-PAGE gel showing the effect of calcium on trypsin digestion of Apaf-1ΔC. Note the persistence of the 40 kDa fragment only in the presence of calcium. (C) Representative SDS-PAGE gels showing the effect of calcium on elastase digestion (left) and chymotrypsin digestion (right) of Apaf-1ΔC. A serial titration of protease concentration was employed to examine the digestion pattern. Note the persistence of a 50 kDa fragment by elastase digestion only in the presence of calcium. Two fragments, at 40 kDa and 24 kDa, are present by chymotrypsin digestion only in the presence of calcium. Molecular Cell 2007 25, 181-192DOI: (10.1016/j.molcel.2006.12.013) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 6 A Schematic Diagram Illustrating the Regulation of Apoptosome Assembly Prior to cytochrome c binding, Apaf-1 exists in an autoinhibited conformation, in which ADP/dADP is bound. Cytochrome c binding is thought to partially relieve the autoinhibition exerted by the WD40 repeats. Subsequent exchange of ADP/dADP for ATP/dATP in Apaf-1 results in the complete removal of autoinhibition and allows it to form an apoptosome. Calcium blocks the exchange of ADP/dADP for ATP/dATP in Apaf-1 and hence inhibits the formation of the apoptosome. Molecular Cell 2007 25, 181-192DOI: (10.1016/j.molcel.2006.12.013) Copyright © 2007 Elsevier Inc. Terms and Conditions